1. Field of the Invention
The present invention is related to the biochemical arts, in particular to therapeutic peptide conjugates.
2. Discussion of the Related Art
The calcitonin (CT) superfamily of peptides includes at least five known members: CT, amylin (AMY), adrenomedullin (ADM), and two calcitonin gene-related peptides, CGRP1 (also known as αCGRP) and CGRP2 (also known as βCGRP). Calcitonin is involved in the control of bone metabolism and is also active in the central nervous system (CNS). Amylin also has specific binding sites in the CNS and is thought to regulate gastric emptying and have a role in carbohydrate metabolism. ADM is a potent vasodilator. ADM has specific receptors on astrocytes and its messenger RNA is upregulated in CNS tissues that are subject to ischaemia. (Zimmermann, et al., Identification of adrenoinedullin receptors in cultured rat astrocytes and in neuroblastoina glioma hybrid cells (NG108-15), Brain Res., 724:238-245 (1996); Wang et al., Discovery of adrenoinedullin in rat ischaemic cortex and evidence for its role in exacerbating focal brain ischaemic damage, Proc. Natl. Acad. Sci. USA, 92:11480-11484 (1995)). The biological activities of CGRP include the regulation of neuromuscular junctions, of antigen presentation within the immune system, of vascular tone and of sensory neurotransinission. (Poyner, D. R., Calcitonin gene-related peptide: multiple actions, multiple receptors, Pharmacol. Ther., 56:23-51 (1992); Muff et al., Calcitonin, calcitonin gene related peptide, adrenomedullin and amylin: homologous peptides, separate receptors and overlapping biological actions, Eur. J. Endocrinol., 133: 17-20 (1995)). Three calcitonin receptor stimulating peptides (CRSPs) have also recently been identified in a number of mammalian species; the CRSPs may form a new subfamily in the CGRP family, however, the endogenous molecular forms, receptors, and biological activity of the CRSPs remain unidentified. (Katafuchi, T and Minamino, N, Structure and biological properties of three calcitonin receptor-stimulating peptides, novel members of the calcitonin gene-related peptide family, Peptides, 25(11):2039-2045 (2004)).
The CT superfamily peptides act through seven-transmembrane-domain G-protein-coupled receptors (GPCRs). The CT receptor and CGRP receptors are type II (“family B”) GPCRs, which family includes other GPCRs that recognize regulatory peptides such as secretin, glucagon and vasoactive intestinal polypeptide (VIP). The best characterized splice variants of human CT receptor differ depending on the presence (formerly CTRII+ or CTR1, now known as CT(b)) or absence (the major splice variant, formerly CTRII− or CTR2, now known as CT(a)) of 16 amino acids in the first intracellular loop. (Gorn et al., Expression of two human skeletal calcitonin receptor isoforms cloned from a giant cell tumor of bone: the first intracellular domain modulates ligand binding and signal transduction, J. Clin. Invest., 95:2680-2691 (1995); Hay et al., Amylin receptors: molecular composition and pharmacology, Biochem. Soc. Trans., 32:865-867 (2004); Poyner et al., 2002). The existence of at least two CGRP receptor subtypes had been proposed from differential antagonist affinities and agonist potencies in a variety of in vivo and in vitro bioassays. (Dennis et al., CGRP8-37, A calcitonin gene-related peptide antagonist revealing calcitonin gene-related peptide receptor heterogeneity in brain and periphery, J. Pharmacol. Exp. Ther., 254:123-128 (1990); Dennis et al., Structure-activity profile of calcitonin gene-related peptide in peripheral and brain tissues. Evidence for receptor multiplicity, J. Pharmacol. Exp. Ther., 251:718-725 (1989); Dumont et al., A potent and selective CGRP2 agonist, [Cys(Et)2,7]hCGRP: comparison in prototypical CGRP1 and CGRP2 in vitro assays, Can. J. Physiol. Pharmacol., 75:671-676 (1997)).
The CGRP1 receptor subtype was found to be sensitive to the antagonist fragment CGRP(8-37). (Chiba et al., Calcitonin gene-related peptide receptor antagonist human CGRP-(8-37), Am. J. Physiol., 256:E331-E335 (1989); Dennis et al. (1990); Mimeault et al., Comparative affinities and antagonistic potencies of various human calcitonin gene-related peptide fragments on calcitonin gene-related peptide receptors in brain and periphery, J. Pharmacol. Exp. Ther., 258:1084-1090 (1991)). By contrast, the CGRP2 receptor was sensitive to linear human CGRP (hCGRP) analogs, in which the cysteine residues at positions 2 and 7 were derivatized (e.g., with acetoaminomethyl [Cys(ACM)2,7] or ethylamide [Cys(Et)2,7]) but CGRP2 receptor was insensitive to fragment CGRP(8-37). (Dennis et al. (1989); Dennis et al. (1990); Dumont et al. (1997)). In 1998, the CGRP1 receptor was identified as a heterodimer composed of a novel single transmembrane domain accessory protein, receptor activity-modifying protein 1 (RAMP1), and calcitonin receptor-like receptor (CRLR or “CL”). (McLatchie et al., RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor, Nature, 393:333-339 (1998)).
CRLR has 55% overall amino acid sequence identity with CT receptor, although the transmembrane domains are almost 80% identical. (McLatchie et al. (1998); Poyner et al., International union of pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin and calcitonin receptors, Pharmacol. Rev., 54:233-246 (2002)).
Ligand specificity of CT receptor and CRLR depend on the coexpression of members of a family of accessory proteins called the receptor activity modifying proteins (RAMPs). The RAMP family includes three that act as receptor modulators that determine the ligand specificity of receptors for the CT family members. RAMPs are type I transmembrane proteins that share about 30% amino acid sequence identity and a common predicted topology, with short cytoplasmic C-termini, one trans-membrane domain and large extracellular N-termini that are responsible for the specificity. (McLatchie et al. (1998); Fraser et al., The amino terminus of receptor activity modifying proteins is a critical determinant of glycosylation state and ligand binding of calcitonin receptor-like receptor, Molecular Pharmacology, 55:1054-1059 (1999)).
CRLR has been shown to form a high affinity receptor for CGRP, when associated with RAMP1, or, to preferentially bind ADM when associated with RAMP2 or RAMP3. (McLatchie et al. (1998); Sexton et al., Receptor activity modifying proteins, Cellular Signaling, 13:73-83 (2001); Conner et al., Interaction of calcitonin-gene-related peptide with its receptors, Biochemical Society Transactions 30(Part 4): 451-454 (2002)). The glycosylation state of CRLR is associated with its pharmacology. RAMPs 1, 2, and 3 transport CRLR to the plasma membrane with similar efficiencies, however RAMP1 presents CRLR as a terminally glycosylated, mature glycoprotein and a CGRP receptor, whereas RAMPs 2 and 3 present CRLR as an immature, core glycosylated ADM receptor. (Fraser et al. (1999)). Characterization of the CRLR/RAMP2 and CRLR/RAMP3 receptors in HEK293T cells by radioligand binding (125I-ADM as radioligand), functional assay (cAMP measurement), or biochemical analysis (SDS-polyacrylamide gel electrophoresis) revealed them to be indistinguishable, even though RAMPs 2 and 3 share only 30% amino acid sequence identity. (Fraser et al. 1999)). Differences have been observed, however, in the pharmacology for CRLR expressed with RAMP 2 versus RAMP 3. Both αCGRP and CGRP8-37 as well as ADM and ADM 22-52 are active at the RAMP 3 heterodimer, indicating that this complex may act as both a CGRP and an ADM receptor. (Howitt et al., British Journal of Pharmacology, 140:477-486 (2003); Muff et al., Hypertens. Res., 26:S3-S8 (2003)). Coexpression of human CRLR with rat RAMP1, and vice versa, showed that the RAMP1 species determined the pharmacological characteristics of the CRLR/RAMP1 complex with respect to several small molecule CGRP receptor antagonists tested. (Mallee et al., Receptor Activity-Modifying Protein 1 determines the species selectivity of non-peptide CGRP receptor antagonists, J. Biol. Chem., 277(16):14294-14298 (2002)). Unless associated with a RAMP, CRLR is not known to bind any endogenous ligand; it is currently the only GPCR thought to behave this way. (Conner et al., A key role for transmembrane prolines in calcitonin receptor-like agonist binding and signaling: implications for family B G-protein-coupled receptors, Molec. Pharmacol., 67(1):20-31 (2005)).
CT receptor has also been demonstrated to form heterodimeric complexes with RAMPs, which are known as amylin receptors. Generally, CT/RAMP1 (AMY1) receptors have high affinity for salmon CT, AMY and CGRP and lower affinity for mammalian CTs. For CT/RAMP2 (AMY2) receptors and CT/RAMP3 (AMY3) receptors, a similar pattern is principally observed, although the affinity for CGRP is lower and may not be significant at physiologically relevant ligand concentrations. The precise receptor phenotype is dependent on cell type and CT receptor splice variant (CT(a) or CT(b)), particularly for RAMP2-generated AMY receptors. For example, a pure population of osteoclast-like cells reportedly expressed RAMP2, CT receptor, and CRLR, but not RAMP1 or RAMP3. (Hay et al. (2004); Christopoulos et al., Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product, Molecular Pharmacology, 56:235-242 (1999); Muff et al., An amylin receptor is revealed following co-transfection of a calcitonin receptor with receptor activity modifying proteins-1 or -3, Endocrinology, 140:2924-2927 (1999); Sexton et al. (2001); Leuthäuser et al., Receptor-activity-modifying protein 1 forms heterodimers with two G-protein-coupled receptors to define ligand recognition, Biochem. J., 351:347-351 (2000); Tilakaratne et al., Amylin receptor phenotypes derived from human calcitonin receptor/RAMP coexpression exhibit pharmacological differences dependent on receptor isoform and host cell environment, J. Pharmacol. Exp. Ther., 294:61-72 (2000); Nakamura et al., Osteoclast-like cells express receptor activity modifying protein 2: application of laser capture microdissection, J. Molec. Endocrinol., 34:257-261 (2005)).
The CGRP-sensitive responses mediated via CT/RAMPs are blocked by the selective receptor antagonist calcitonin(8-32) but not by CGRP(8-37), which antagonizes the CRLR/RAMP1 complex (CGRP1 receptor). (Kuwasako et al., Novel calcitonin-(8-32)-sensitive adrenomedullin receptors derived from co-expression of calcitonin receptor with receptor activity-modifying proteins, Biochem. Biophys. Res. Commun., 301:460-464 (2003); Leuthäuser et al., 2000).
The αCGRP peptide (also known as CGRP1) and (βCGRP peptide (also known as CGRP2) are 37 amino acid residues long and differ from each other by three amino acids. These two isoforms have so far proved to be indistinguishable in their biological activities. (Poyner, D. R. (1992); Muff et al. (1995)). Native human αCGRP and βCGRP each contain a disulfide bridge between cysteine residues at amino acid positions 2 and 7 and a carboxy-terminal phenylalanine amide, both of which are required for biological activity of the native peptides. (Wimalawansa, S J, Amylin, calcitonin gene-related peptide, calcitonin and adrenomedullin: a peptide superfamily, Crit. Rev. Neurobiol., 11: 167-239 (1997)). Their binding sites are widely distributed among peripheral tissues and in the central nervous system, enabling CGRP to exert a wide variety of biological effects, including potent vasodilation. (Van Rossum et al., Neuroanatomical localization, pharmacological characterization and functions of CGRP, related peptides and their receptors, Neurosci. Biobehav. Rev., 21:649-678 (1997)).
Structural studies investigating the interaction of CGRP with its receptor have defined both functional sub-domains and specific residues involved in receptor binding and activation. (Conner, A. C., Biochem. Soc. Trans., 30:451-455 (2002)). As illustrated in FIG. 1, the first seven amino acid residues of CGRP, which form a disulphide-bonded loop, are thought to interact with the transmembrane domain of CRLR to cause receptor activation. CGRP(8-37) fragment binds with high affinity to CGRP1 receptor, but acts as an antagonist. The rest of the CGRP molecule falls into three domains or regions: amino acid residues 28-37 and 8-18 are normally required for high-affinity binding with the CGRP1 receptor, while residues 19-27 form a hinge region, which appears to allow a hairpin-like structure wherein the two sub domains interact with one another at the receptor interface. The 28-37 region is thought to be in direct contact with the receptor during binding, while the α-helical region comprising residues 8-18 may make additional receptor contacts or may stabilize an appropriate conformation of the 28-37 region. It is likely that these binding regions of CGRP interact with CGRP1 receptor both at the CRLR and at the extracellular domain of RAMP1. The amidated carboxy-terminal residue has been shown to be essential for high affinity binding to CGRP1 receptors by CGRP and CGRP peptide analogs. Mutation analyses have further implicated select residues R11, R18, T30, V32 S34 and F37 in receptor binding. (Rist et al., From micromolar to nanomolar affinity: a systematic approach to identify the binding site of CGRP at the human calcitonin gene-related peptide 1 receptor, J. Med. Chem., 41:117-123 (1998); Conner et al., Interaction of calcitonin-gene-related peptide with its receptors, Biochem. Soc. Trans., 30(4):451-455 (2002); Smith et al., Modifications to the N-terminus but not the C-terminus of calcitonin gene-related peptide(8-37) produce antagonists with increased affinity, J. Med. Chem., 46:2427-2435 (2003)).
Moreover, amino-terminal truncations of CGRP (e.g., CGRP(8-37)) have been identified that are antagonistic to the receptor. While further truncations (e.g., CGRP(28-37)) show very little receptor binding, much of the lost antagonist activity can be restored with three point mutations (T30D, V32P and G33F) in the CGRP(28-37) region. (Rist, B. et al., J. Med. Chem., 41:117-123 (1998)). It has been suggested that these amino acid substitutions compensate for the missing (CGRP(8-27) domain (Carpenter, K. A. et al., Turn structures in CGRP C-terminal analogues promote stable arrangements of key residue side chains, Biochemistry, 40:8317-8325 (2001)).
CGRP is thought to have a causative role in migraine. Migraine pathophysiology involves the activation of the trigeminal ganglia, where CGRP is localized, and CGRP levels significantly increase during a migraine attack. This in turn, promotes cranial blood vessel dilation and neurogenic inflammation and sensitization. (Doods, H., Curr. Opin. Investig. Drugs, 2:1261-1268 (2001)). CGRP has been shown to induce migraine headaches in patients susceptible to migraines. Furthermore, in a recent Phase II clinical trial, a potent small-molecule CGRP antagonist has been shown to alleviate migraine pain. CGRP may also be involved in chronic pain syndromes other than migraine. In rodents, intrathecally delivered CGRP induces severe pain, and CGRP levels are enhanced in a number of pain models. In addition, CGRP antagonists block neuropathic and capsaicin-induced pain in rodents. Together, these observations imply that a potent and selective CGRP receptor antagonist can be an effective therapeutic for treatment of chronic pain, including migraine.
CGRP has also been implicated in diabetes mellitus (type II), inflammation, cardiovascular disorders, and in the hemodynamic derangements associated with endotoxemia and sepsis resulting from postoperative infection and a variety of other infectious diseases. (E.g., Khachatryan, A et al., Targeted expression of the neuropeptide calcitonin gene-related peptide to beta cells prevents diabetes in NOD mice, J. Immunol., 158(3):1409-1416 (1997); Ohtori S et al., Phenotypic inflammation switch in rats shown by calcitonin gene-related peptide immunoreactive dorsal root ganglion neurons innervating the lumbar facet joints, Spine, 26(9):1009-1013 (2001); Qin, X et al. Temporal and spatial distribution of Substance P and its receptor regulated by calcitonin gene-related peptide in the development of airway hyperresponsiveness, FEBS Journal, 30th FEBS Congress & 9th IUMB Conference Website, Abstract No. M3-020P (2005); Brain, S D et al., Evidence that calcitonin gene-related peptide contributes to inflammation in the skin and joint, Ann. NY Acad. Sci., 657(1):412-419 (1992); Caviedes-Bucheli, J. et al., Expression of calcitonin gene-related peptide(CGRP) in irreversible acute pulpitis, J. Endodontics, 30(4):201-204 (2004); Ling, Q D et al., The pattern and distribution of calcitonin gene-related peptide (CGRP) terminals in the rat dorsal following neonatal peripheral inflammation, Neuroreport, 14(15):1919-1921 (2003); Li, Y J et al., CGRP-mediated cardiovascular effect of nitroglycerin, Med Hypotheses, 60(5):693-698 (2003); Beer, S et al., Systemic neuropeptide levels as predictive indicators for lethal outcome in patients with postoperative sepsis, Critical Care Medicine, 30(8):1794-1798 (2002)).
Therapeutic administration of CGRP analogs was taught by Evans et al. for the lowering of blood pressure and gastric acid secretion, and for other effects on, for example, ingestion behavior, taste and sensory perception, e.g., nociception. (U.S. Pat. No. 4,530,838; U.S. Pat. No. 4,736,023).
Therapeutic use of CGRP antagonists and CGRP-targeting aptamers has been proposed for the treatment of migraine and other disorders. (E.g., Olesen et al., Calcitonin gene-related peptide receptor antagonist BIBN 4096 BS for the acute treatment of migraine, New Engl. J. Med., 350:1104-1110 (2004); Perspective: CGRP-receptor antagonists—a fresh approach to migraine, New Engl. J. Med., 350:1075 (2004); Vater et al., Short bioactive Spiegelmers to migraine-associated calitonin gene-related peptide rapidly identified by a novel approach: tailored-SELEX, Nuc. Acids Res., 31(21 e130):1-7 (2003); WO 96/03993).
For example, Noda et al. described the use of CGRP or CGRP derivatives for inhibiting platelet aggregation and for the treatment or prevention of arteriosclerosis or thrombosis. (EP 0385712 B1).
Liu et al. disclosed therapeutic agents that modulate the activity of CT receptor, including vehicle-conjugated peptides such as calcitonin and human αCGRP. (WO 01/83526 A2; US 2002/0090646 A1).
Vasoactive CGRP peptide antagonists and their use in a method for inhibiting CGRP binding to CGRP receptors were disclosed by Smith et al.; such CGRP peptide antagonists were shown to inhibit CGRP binding to coronary artery membranes and to relax capsaicin-treated pig coronary arteries. (U.S. Pat. No. 6,268,474 B1; and U.S. Pat. No. 6,756,205 B2).
Rist et al. disclosed peptide analogs with CGRP receptor antagonist activity and their use in a drug for treatment and prophylaxis of a variety of disorders. (DE 19732944 A1).
Nevertheless, CGRP peptides have shown a number of problems as therapeutics. Native CGRP peptides are typically nonselective, inactive in oral form, generally have a short duration of action and can elicit a number of potential side effects that can include undesirable effects on blood pressure. In general, therapeutic peptides and proteins exhibit very fast plasma clearance, thus requiring frequent injections to ensure steady pharmaceutically relevant blood levels of a particular peptide or protein with pharmacological activity. Many pharmaceutically relevant peptides and proteins, even those having human primary structure, can be immunogenic, giving rise to neutralizing antibodies circulating in the bloodstream. This is especially true for intravenous and subcutaneous administration, which is of particular concern for the delivery of most peptide and protein drugs.
By increasing the molecular volume and by masking potential epitopes, modification of a therapeutic polypeptide with a vehicle, such as a polyethylene glycol (PEG) polymer, has been shown to be effective in reducing both the rate of clearance as well as the antigenicity of the protein. Reduced proteolysis, increased water solubility, reduced renal clearance, and steric hindrance to receptor-mediated clearance are a number of mechanisms by which the attachment of a polymer to the backbone of a polypeptide may prove beneficial in enhancing the pharmacokinetic properties of the drug. For example, Davis et al. taught conjugating PEG or polypropylene glycol to proteins such as enzymes and insulin to produce a less immunogenic product while retaining a substantial proportion of the biological activity. (U.S. Pat. No. 4,179,337).
In the actual practice of developing a conjugated peptide drug, it is not a trivial matter to overcome the significantly lower potency that a conjugated form typically exhibits relative to the unconjugated form of the peptide. (J. M. Harrist et al., PEGylation: A Novel Process for Modifying Pharmacokinetics, Clin. Pharmacokinet., 40:539-551 (2001); and R. Mehvar, Modulation of the Pharmacokinetics and Pharmacodynamics of Proteins by Polyethylene Glycol Conjugation, J. Pharm. Pharmaceut. Sci., 3:125-136 (2000)).
It is, therefore, a desideratum to combine the therapeutic benefits of vehicle-conjugated CGRP peptides and analogs (particularly, but not limited to, those with CGRP receptor antagonist activity), such as substantially increased pharmacological half-life and decreased immunogenicity, with little, if any, loss of potency relative to unconjugated forms. These and other benefits are provided by the present invention.